The effect of ethanol on glial cell functions has been recently identified by the Division of Neuroscience and Behavior of the National Institute of Alcohol Abuse and Alcoholism as a priority scientific area. This proposal focuses on the role of astrocytes as mediators of the teratogenic effect of ethanol on the central nervous system. Astrocytes are able to modulate neuritogenesis and synaptogenesis by secreting neuroactive proteins and peptides and by regulating the composition of the extracellular matrix. What controls astrocytes secretion is still unknown. We hypothesize that muscarinic receptor stimulation in astrocytes by neuron- derived acetylcholine shifts astrocyte secretion toward a permissive environment for neuronal differentiation. We have indeed observed that muscarinic receptor stimulation in astrocytes induces neuritogenesis and synaptogenesis in hippocampal neurons co-cultured with astrocytes or exposed to astrocyte conditioned medium. We also found that ethanol inhibits this effect, thus revealing a potentially new and important mechanism contributing to the deleterious effects of alcohol on fetal brain. The overarching hypothesis of this proposal is that the inhibition by ethanol of muscarinic signaling in astrocytes, which has been characterized during the previous funding period, would hamper their ability to stimulate neuronal differentiation, by affecting astrocyte secretion. The specific aims of this proposal are: 1: To characterize the intracellular mechanisms involved in carbachol-treated astrocyte promotion of neuritogenesis and synaptogenesis in hippocampal neurons by means of biochemical, immunocytochemical and morphological techniques, and to assess the inhibitory effect of ethanol on this action. 2: To investigate factors released by carbachol- and/or- ethanol-treated astrocytes using a quantitative proteomic approach. 3: To investigate the effect of carbachol and ethanol on the transcription and release by astrocytes of fibronectin, laminin, plasminogen activator inhibitor 1, thrombospondin 1, neurocan and brevican; these factors are released by astrocytes, and have been shown, in preliminary experiments, to be affected by carbachol or ethanol. In addition, the role of all these factors on hippocampal neuron differentiation will be investigated by silencing their expression or by selectively removing them from astrocyte-conditioned medium. 4: To substantiate our observations of astrocyte-neuron interactions in isolated cell preparations with experiments carried out in a more complex system, the hippocampal slice. Altogether, these studies are aimed at elucidating novel aspects of the communication between astrocytes and neurons and at characterizing new mechanisms involved in the developmental neurotoxicity of ethanol.